Faulty regulation of apoptosis and the cell cycle are critical determinants of cancer pathogenesis. These essential cellular processes are modulated, in health and disease, by multiple protein networks. Among these, the BCL-2 family of pro- and anti-apoptotic proteins, as well as the p53 tumor suppressor protein, stand out due to their essential roles in triggering apoptosis and cell cycle arrest in response to diverse stimuli. Newly identified interactions between p53 and select BCL-2 members, such as BAX and BCL-XL, impact the survival of cells, and these interactions are dictated by the three-dimensional molecular structure of specific peptide domains located within their protein architecture. Defects in these interactions lead to pathologic deregulation of apoptotic and growth arrest signaling pathways. The goal of this research is to use a multidisciplinary approach to target the control points of the integrated BCL2-family-p53 apoptotic signaling pathway in an effort to investigate and reactivate apoptosis in cancer cells. To that end, we propose to: 1. Synthesize diversified hydrocarbon-stapled peptide libraries of BID BH3, PUMA BH3, and p53 and evaluate their biochemical and biophysical properties;2. Optimize the intracellular delivery and versatility of stapled peptides through chemical derivatization;3. Evaluate the ability of stapled BID BH3, PUMA BH3, and p53 peptides, singly and in combination, to induce cell cycle arrest and/or activate BAX-induced mitochondrial apoptosis in vitro and in murine xenograft models. Peptide helices that engage protein binding pockets hold promise as a novel set of chemical tools to study and treat cancer. Our initial panels of BID BH3 and p53 stapled peptides demonstrate striking biochemical and pharmacologic properties, including enhanced (-helicity, protease resistance, and cell permeability compared to the corresponding unmodified peptides. Importantly, the stapled peptides recapitulate the biological activity of the native protein domains in vitro and in vivo. The recently identified intersection of the BCL-2 family and p53 pathways at the BAX control point of apoptosis opens new opportunities to develop next-generation stapled peptides to induce cell cycle arrest synergistically and reactivate apoptosis in cancer cells. The research proposed here aims to apply novel synthetic approaches such as hydrocarbon-stapling to reinforce natural peptide ligands relevant to deregulated BCL-2 and p53 pathways, thereby providing alternative tool compounds to study and manipulate protein interactions relevant to the pathogenesis and maintenance of cancer. A multidisciplinary group of faculty mentors, collaborators, and academic advisors with expertise spanning organic chemistry, structural biology, cancer biology, and clinical oncology will provide an ideal environment for Dr. Bernal's development as an independent investigator in the field of cancer chemical biology.